M11L forms a complex with the PBR. THP-1 cells were infected with VVM11L or VV601. Protein within hypotonic cell lysates was cross-linked with DSP, extracted with an NP-40/deoxycholate buffer, and immunoprecipitated with an anti-M11L (A and B) or anti-PBR (C and D) antibody. Samples subjected to cross-linker cleavage (+) or left uncleaved (−) were analyzed by SDS-PAGE and immunoblotting with either an anti-M11L (A and C) or anti-PBR (B and D) antibody. M11L immunoprecipitated from cells infected with VVM11L in the absence of cross-linking or a THP-1 cell whole cell lysate (WCL) were included as controls.

Stable expression of M11L in Rat2 cells provides protection against staurosporine- and PPIX-mediated cytochrome c release. (A) Rat2puro and M11L-expressing Rat2M11L cells were treated with staurosporine (+STS) or DMSO solvent alone (−STS) as control. Cytochrome c redistribution into the cytosolic fraction of cell lysates was detected by SDS-PAGE and immunoblot analysis (top panel). Equivalent amounts of protein were analyzed as shown by the actin immunoblot (bottom panel). (B) Treatment of permeabilized cells with 20 μM PPIX induced greater cytochrome c redistribution in Rat2puro cells than Rat2M11L cells (top panel) when equivalent amounts of the cytosolic fraction were loaded as assessed by actin content (bottom panel).

PBR expression augments the protective effects of myxoma virus infection. Jurkat or Jurkat-PBR cells were mock-infected (mock) or infected with myxoma virus (Myx) or the M11L knockout virus (M11L⁻). The percentage of cells that underwent a loss of TMRE fluorescence as a result of staurosporine treatment (A) or Fas stimulation (B) is shown. Results are the average of three independent experiments (±SD). In parallel, Jurkat or Jurkat-PBR cells that had been mock- or virus-infected were analyzed for the presence of M11L using immunoprecipitation and immunoblotting (C, top panel). Alternatively, virus infection was verified by the production of the early viral protein, M-T7 (C, bottom panel).

M11L can be cross-linked into a complex (A). Cos-7 cells were infected with the M11L-expressing vaccinia virus VVM11L or the control virus VV601 and labeled with ³⁵S-methionine/cysteine. Proteins present in hypotonic cell lysates were cross-linked with the hydrophobic cross-linker DSS (+DSS) or left uncrosslinked (−DSS), before extraction with an NP-40/deoxycholate buffer, immunoprecipitation with anti-M11L antibody and analysis by SDS-PAGE and autoradiography. (B) THP-1 and HeLa cells infected with VVM11L or VV601 were subjected to hypotonic lysis and DSS cross-linking. M11L was immunoprecipitated as described and detected by SDS-PAGE followed by immunoblot analysis. (C) BGMK cells were infected with the vMyxlac parental myxoma virus (Myx) or the M11L knockout strain vMyxM11L⁻ (M11L⁻) and again samples were subjected to hypotonic lysis, DSS cross-linking, and immunoprecipitation and analysis of M11L.

EYFP-M11L and fluorescently labeled PBR undergo FRET (A). Confocal image of a representative HeLa cell monolayer stained with FGIN-1–27-NBD (shown in green pseudocolor) acquired using a 548 nm excitation line and emission filter of 462–537 nm before photobleaching. (B) EYFP-M11L expression in the same monolayer (shown in red pseudocolor) acquired using a 515 nm excitation line and emission filter of 526–719 nm before photobleaching. (C) Unmixed signal obtained using a 458 nm laser line excitation. The cells marked with an asterisk were photobleached. Regions of interest were drawn over the cells to be analyzed. Here, regions designated by red and green are a pair of photobleached cells and regions designated by blue and yellow are an unbleached pair of cells. All cells are stained with FGIN-1–27-NBD and the cells designated by red and blue outlines express EYFP-M11L. (D) DIC image of the field of view showing cell morphology. Scale bar: 10 μm. (E) Intensity of EYFP–expressing cell (red region in C) before and after photobleaching. Notice the dequenching of NBD signal after photobleaching (arrow). (F) Intensity of the non-EYFP expressing cell (green in C) before and after photobleaching (arrow). (G) Averaged normalized intensity changes before and after photobleaching in 38 fields of cells analyzed (standard error, P < 0.0000000233 and 0.0000000186 for time points 4 and 5 respectively). The y axis represents the intensity variation of the FGIN-1-27-NBD (donor) channel. The x axis represents the sequence of time lapse points. Arrow indicates the bleaching point.

The mitochondrial targeting of M11L is important for inhibition of ΔΨ_m loss induced by the PBR ligands PK11195 and PPIX. (A) Cells expressing EGFP appended with truncated, nontargeted M11L (stop), full length M11L, the mitochondrial targeting sequence (mt), or Bcl-2 were treated with 5 μM staurosporine (STS) or 5 μM staurosporine in combination with 120 μM PK11195 (STS+PK1195). The reduction in the percentage of TMRE-positive EGFP-expressing cells after treatment is shown. The data represent the average of three independent experiments (±SD). (B) Cells expressing EGFP chimeras were treated with 25 or 30 μM PPIX. The reduction in the percentage of TMRE positive EGFP-expressing cells after treatment is shown. The results represent the average of three independent experiments (±SD).